| Mosquitoes not only suck blood,but also spread a variety of diseases,such as malaria,dengue fever,filariasis,Zika,Japanese encephalitis and so on.Mosquito-borne diseases pose a huge threat to human health in worldwide.Because most mosquito-borne diseases do not have vaccines and targeted drugs,controlling their vectors has always been the most important measure to prevent and control mosquito-borne diseases,of which chemical control is the main one.With the long-term and large-scale use of chemical insecticides,mosquito resistance has become a more and more serious problem.Researchers have begun to seek new technical methods for mosquito control.The olfactory system of mosquitoes participates in important life activities such as mating,blood-sucking,and egg-laying.Studying the mechanism of mosquito's olfactory behavior can provide a theoretical basis for the development of new mosquito control methods and blocking the blood-sucking behavior of mosquitoes to block the transmission of mosquito-borne diseases.In this study,the olfactory behavior mechanism of Culex pipiens quinquefasciatus,an important vector mosquito in the world,was studied A new set of mosquito olfactory wind tunnel was developed to test the olfactory behavior of mosquitoes.Through transcriptome sequencing and bioinformatics analysis of the olfactory tissues of Cx. pipiens quinquefasciatus and Cx.pipiens molestus both belonging to the Culex pipiens Complex before and after blood sucking,the olfactory genes with expression differences were verified by quantitative PCR analysis and screened out for olfactory genes that may be related to the blood-sucking behavior of Cx.pipiens quinquefasciatus.RNAi technology was used to verify the blood-sucking behavior of the screened target olfactory genes.The main results of this study include:1.The newly developed mosquito olfactory wind tunnel is mainly divided into two parts: the airway control system and the behavior screening system.Through the test of different temperature,humidity,wind speed and other environmental factors,the results show that the temperature 30 how the humidity 40 h and the wind speed0.2m/s is the best factor for olfactory wind tunnel to be applied to mosquito olfactory behavior test.When nothing is used,the attracting rate of the mouse to Cx.pipiens quinquefasciatus was 35.6±5.1%,the attracting rate of the arm to Aedes aegypti is44.1±23.1%,which was significantly higher than that of the blank control(P<0.05). When four kinds of repellents were used,the attracting rates of Cx.pipiens quinquefasciatus were 0%,0%,3.8±3.3% and 3.8±1.6%,respectively,and the attracting rates of Ae.aegypti were 4.4±0.4%,2.7±0.2%,3.6±1.2% and 3.6±1.2%,respectively.Compared with the control group,the ability of arm and mouse to attract mosquitoes was significantly reduced when the repellent was used,and there was a significant difference(P<0.05).The test results show that the air flow in the wind tunnel is stable,and the air flow in the control tube and the experimental tube flows in one direction without interfering with each other.The wind tunnel can be widely used in various olfactory behavior tests of mosquitoes.2.Transcriptome sequencing and bioinformatics analysis of the olfactory tissues of Cx.pipiens quinquefasciatus and Cx.pipiens molestus before and after blood-sucking showed that there were a total of 6690 genes with significant differences in expression after blood-sucking Cx.pipiens quinquefasciatus and Cx.pipiens molestus.The genes were significantly up-regulated and down-regulated accounted for 14.68% and18.71%,respectively.After the Cx.pipiens molestus sucked blood,there were a total of 7654 genes with significant differences in expression,and the significantly up-regulated and down-regulated genes accounted for 17.56% and 20.68%,respectively.In terms of olfactory genes,a total of 57 OBP genes and 94 OR genes were annotated in the antennal antennae transcriptome of Cx.pipiens quinquefasciatus before and after blood sucking,and a total of 60 OBP genes and 77 OR genes were annotated in the olfactory tissue transcriptome of Cx.pipiens quinquefasciatus before and after blood sucking.Compared with before blood-sucking,1 OR gene and 16 OBP genes were up-regulated in Cx.pipiens quinquefasciatus and Cx.pipiens molestus,and 6 OR genes were down-regulated together.The number of OBP genes that were significantly up-regulated and down-regulated in Cx.pipiens quinquefasciatus after sucking blood was 1 and 18 respectively.The number of OBP genes that were significantly up-regulated and down-regulated after sucking Cx.pipiens quinquefasciatus compared with before blood-sucking were 2 and 19.There were 9 OR genes that were significantly up-regulated and down-regulated after the mosquitoes sucked blood compared to before sucking blood.The OR genes that were significantly up-regulated and down-regulated after sucking Cx.pipiens quinquefasciatus were 3 and 14 respectively.The results of protein interaction network analysis show that there are many interactions between several OR genes and OBP genes.It is speculated that they can ensure the stability of gene family functions through gene compensation,so as to ensure the smooth progress of mosquitoes' olfactory behavior.3.The olfactory genes selected by the transcriptome sequencing analysis were verified by real-time fluorescent quantitative PCR.The results showed that the Cx. pipiens quinquefasciatus after blood sucking significantly up-regulated 2 OBP genes,5 OR genes and 4 OBP genes were significantly down-regulated compared to before blood sucking.After Cx.pipiens molestus after blood sucking,1 OR gene was significantly up-regulated,and 5 OR genes and 5 OBP genes were significantly down-regulated compared to before blood-sucking.The PCR verification results were consistent with the sequencing data.Based on the results of transcriptome sequencing and quantitative PCR analysis,four olfactory genes,OR1,OR5,OR78,and OR83,were selected to apply RNAi method to verify the blood-sucking behavior function.After ds RNA microinjection of the female Cx.pipiens quinquefasciatus,the results showed that the blood-sucking rates of the female Cx.pipiens quinquefasciatus were significantly lower than the blood sucking rate of the group/negative control group was 100%/70%,100%/50% and 100%/50% and(P<0.05).The blood sucking rate after OR83-ds RNA injection was 36.36%,which was significantly lower than that of the blank control group 100%,but there is no statistical difference in the blood-sucking rate compared with the negative control group.It is speculated that OR1,OR5 and OR78 genes are related to the blood-sucking behavior of Cx.pipiens quinquefasciatus.Whether OR83 gene is related to the blood-sucking behavior of mosquitoes needs further experimental research to verify. |
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